1. Field of the Invention
The present invention relates generally to analyzing an enzyme solution. More particularly, the invention relates to facilitating an activity determination for gel-based/chromatographic-based endpoint or dose response analysis.
2. Related Art
Electrophoresis is a technique used to separate and analyze single charged molecules. The charged molecules can be placed on any type of support matrix, such as paper, cellulose acetate, starch gel, agarose gel, or acrylamide gel. Generally, a buffer is run in a separation medium containing the support matrix, and an electric field is applied to the support matrix. At the end of the run, the support matrix is stained appropriately for visualization of the molecules within the matrix.
Since agarose and acrylamide gels are porous substances, an agarose or acrylamide gel-based separation medium permits the molecules to be separated by size or molecular weights. The gels, therefore, retard or prevent larger molecules from moving, and allow the smaller molecules to migrate freely. Agarose gels are generally used to separate larger macromolecules, such as, nucleic acids, large proteins and protein complexes, because agarose gels tend to be more rigid and easy to handle. Acrylamide gels, on the other hand, are more commonly used to separate medium or smaller-sized proteins and small oligonucleotides requiring a smaller gel pore size for retardation.
Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), tend to carry a negative charge in any buffer used for electrophoresis. As such, nucleic acids have a propensity to separate according to their molecular weight. The electrophoretic separation of a protein, however, is based on its electrical charge and molecular weight. Since proteins are amphoteric compounds, their electrical charge depends on the pH of the buffer used for electrophoresis. If the pH exceeds the protein""s isoelectric point, a negative charged protein would migrate towards the anode in the electrical field. If the pH is below the isoelectric point, the protein is likely to have a positive charge causing it to migrate towards the cathode.
A restriction endonuclease (i.e., restriction enzyme) is added to cleave a nucleic acid (e.g., DNA or RNA) at certain sites along the macromolecule. Similarly, a proteinase or protease (i.e., proteolytic enzyme) is used to break protein chains into shorter peptides or break the peptides into amino acids.
With respect to DNA, a restriction enzyme has the ability to recognize a short, specific sequence of nucleotide bases (such as, adenine, cytosine, thymine, and guanine) and severe the DNA molecule at these recognition sites by catalyzing the hydrolysis of the bond between adjacent nucleotides. Although some types of restriction enzymes are known to cleave DNA at specific sites within the recognition site; other types of restriction enzymes cleave DNA randomly, sometimes hundreds of bases from the recognition sequence.
A restriction enzyme""s ability to cut DNA at precise locations is germaine to a researcher""s ability to isolate gene fragments and recombine them with other DNA molecules. Understandably, precise manipulation of DNA fragments is crucial to recombinant DNA technology or genetic engineering.
It is also important to be able to accurately measure or determine the unit activity of a restriction enzyme. The unit activity (also referred to as the xe2x80x9cunit callxe2x80x9d) is the least concentrated dilution of restriction enzyme (specifically, proteolytic enzyme for proteins) that results in a complete digestion of the macromolecule (i.e., nucleic acid or protein).
Conventionally, one may determine the unit activity by visually detecting when, for example, a DNA fragment would disappear into the background of a support matrix. Such subjective calls are prone to human error and inherent inaccuracies. As a result, the unit activity could be misjudged by a significant factor. Subjective quantitation of enzymes makes it difficult to produce consistent products and control production costs.
Another problem is related to the separation medium used to analyze DNA fragments. The separation medium may have multiple wells or lanes for apportioning the DNA samples throughout the gel. Each lane represents the result of one reaction. Adjacent lanes can be related to each other such that, as one travels from left to right, each lane represents the result of decreasing concentration of the enzyme used in each reaction to generate the visualized banding pattern seen in each lane of the gel. As the DNA fragments separate during electrophoresis, various factors (such as pH levels) can interfere with the flow in each lane. These factors can prevent each lane from running equivalently. In other words, the fragments do not migrate equivalent distances throughout the gel. This results in a wavy pattern (also known as a smile effect) that makes it difficult to align the DNA fragments across lanes and interpret the electrophoretic results.
Thus, there is a need in the art for a method and device that can accurately and objectively determine the unit activity or other catalytic results of a restriction enzyme.
The present invention is directed to a method and system for processing fragment population information that is generated from a stained macromolecule situated in a separation medium to objectively and quantitatively determine catalytic results (such as, the unit activity) of an enzyme. The term xe2x80x9cenzyme,xe2x80x9d as used herein, is intended to include restriction enzymes, proteolytic enzymes or the like.
A test aliquot, comprising a macromolecule (such as, DNA, RNA, protein, peptide or the like) and diluted enzyme concentration, is distributed in the separation medium containing a plurality of wells. The enzyme concentration acts as a catalysis to cleave the macromolecule into distinct fragments prior to the macromolecule being distributed in the separation medium. Each well within the separation medium produces a distinct lane of electrophoretic results. Adjacent wells, and hence lanes, have relationship with one another. From left to right, each lane represents the result of decreasing concentration of the enzyme used in each reaction to generate a visualized banding pattern seen in each lane. The lane-to-lane dilution difference in enzyme concentration is the same from one lane to the next.
In an embodiment of the present invention, an intensity data profile is produced from digital images of the fragments to produce a series of stacked profiles. The stacked profiles are used to provide a model of the fragments resolved from the electrophoretic separation and residing in lanes below the reaction wells of the separation medium.
The stacked profiles are vertically aligned to designate and assign each fragment within its respective lane aligned to the identical fragment in each adjacent lane. A group of partial band(s) and final band(s) are selected from the fragments. Peak integrations are implemented to measure the intensity of the partial and final bands. A series of intensity ratios are computed from the peak integrations. The ratios embody the intensity of the specified partial band relative to the intensity of the specified final band.
After the intensity ratios have been computed and normalized, the intensity ratios are used to produce a trend approximation. A threshold crossing value is assigned a value at which the trend approximation crosses below a threshold crossing level. This unique intersection is characteristic of the enzyme strength of the original test sample.
A product-specific calibration factor is calculated by dividing the threshold crossing value by a historical unit value. The historical unit value is the industry-specified amount of restriction enzyme required to obtain complete digestion of, for example, a DNA substrate under specified assay conditions. Once the calibration factor has been calculated, the threshold crossing value is divided by the calibration factor to determine the reported unit call.
In an embodiment, a technique is provided to highlight certain data if their removal would change the results by more than one-half of a lane interval, or if data points are unusually far from the fitted model. The operator can review the highlighted points, exclude the data from analysis, and refit the model.